Investigating Pseudouridylation Mechanisms by High-Throughput in Vitro RNA Pseudouridylation and Sequencing
Investigating Pseudouridylation Mechanisms by High-Throughput in Vitro RNA Pseudouridylation and Sequencing
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DOI：
10.1007/978-1-0716-1374-0_22
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发表时间：
2021-01-01
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影响因子：
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通讯作者：
Gilbert, Wendy V.
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文献类型：
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作者：
Martinez, Nicole M.;Gilbert, Wendy V.
假尿苷分析揭示了细胞RNA中许多以前未知的RNA修饰假尿苷（Psi）位点。所有生物体都表达多种假尿苷激酶（PUS），其RNA靶点和靶向机制仍有待阐明。在这里，我们描述了一种高通量的体外假尿苷化测定，以询问假尿苷状态后，孵育与重组假尿苷酶（PUS）在数以千计的RNA序列的利益平行。这种方法允许验证在细胞中暂时鉴定的位点，鉴定个体PUS的直接靶点，以及询问靶识别的决定因素，包括一级序列和RNA二级结构。
Pseudouridine profiling has revealed many previously unknown sites of the RNA modification pseudouridine (Psi) in cellular RNAs. All organisms express multiple pseudouridine synthases (PUS) whose RNA targets and mechanisms of targeting remain to be elucidated. Here, we describe a high-throughput in vitro pseudouridylation assay to interrogate pseudouridine status upon incubation with recombinant pseudouridine synthases (PUS) at thousands of RNA sequences of interest in parallel. This approach allows validation of sites provisionally identified in cells, identification of the direct targets of individual PUS, and interrogation of the determinants of target recognition including primary sequence and RNA secondary structure.